Since the early 1950s, clinical management of problems associated with ovarian insufficiency and failure, including infertility due to aging or insults, has been restricted by the belief that the pool of oocytes set forth at birth is not amenable to replacement or renewal (Zuckerman, Recent Prog Horm Res 1951 6:63-108). In other words, any therapeutic intervention had to conform to manipulation of the existing stockpile of oocyte-containing follicles to produce a desired clinical outcome. In 2004, however, studies with mice challenged the idea of a fixed ovarian reserve of oocytes being endowed at birth (Johnson et al., Nature 2004 428:145-150). Based on results from several experimental approaches, it was concluded that ovaries of adult female mammals retain rare germline or oogonial stem cells (OSCs) that routinely produce new oocytes in a manner analogous to germline stem cell support of sperm production in the adult testis (Spradling, Nature 2004 428:133-134). Several years later, OSCs were successfully isolated from neonatal and adult mouse ovaries (Zou et al., Nat Cell Biol 2009 11:631-636; Pachiarotti et al., Differentiation 2010 79:159-170). Collectively, these investigations, along with several other reports from studies of mice (Johnson et al., Cell 2005 122:303-315; Wang et al., Cell Cycle 2010 9:339-349; Niikura et al., Aging 2010 2:999-1003) have conceptually validated the use of OSCs as agents for transplantation and as targets for new therapies to modulate ovarian function and female fertility (Tilly et al., Biol Reprod 2009 80:2-12; Tilly et al., Mod Hum Reprod 2009; 15:393-398). In addition, the identification of dormant OSCs in atrophic ovaries of aged mice, which spontaneously resume oocyte formation when exposed to a young adult ovarian environment, indicates that ovarian aging may be reversible (Niikura et al., Aging 2009 1:971-978; Massasa et al., Aging 2010; 2:1-2). The clinical utility of OSCs is now further confirmed by evidence shown herein that a comparable population of oocyte-producing stem cells exists in, and can be purified from, ovaries of healthy reproductive-age women.
Although these new studies indicate that oocyte numbers in adult ovaries are amenable to therapeutic expansion through OSC-based technology, ovarian aging and failure is determined by both a decline in oocyte number as well as a decline in the quality of the oocytes present in the ovaries. Hence, it is imperative to identify methods for improving oocyte quality, especially in women of advancing maternal age. During the past few decades, because of cultural and social changes, women in the developed world have significantly delayed childbirth. For example, first birth rates for women 35-44 years of age in the United States have increased by more than 8-fold over the past 40 years (Ventura Vital Health Stat 2009 47:1-27; Matthews, NCHS Data Brief 2009 21:1-8). It is well known that pregnancy rates in women at 35 or more years of age are significantly lower, both naturally and with assisted reproduction. The decline in live birth rate reflects a decline in response to ovarian stimulation by gonadotropin hormones (follicle-stimulating hormone or FSH, and luteinising hormone or LH), reduced oocyte and embryo quality and pregnancy rates, and an increased incidence of miscarriages and fetal aneuploidy. In fact, aging-associated chromosomal and meiotic spindle abnormalities in eggs are considered the major factors responsible for the increased incidence of infertility, fetal loss (miscarriage) and conceptions resulting in birth defects—most notably trisomy 21 or Down syndrome—in women at advanced reproductive ages (Henderson et al., Nature 1968 218:22-28; Hassold et al., Hum Genet 1985 70:11-17; Battaglia et al., Hum Reprod 1996 11:2217-2222; Hunt et al., Trends Genet 2008 24:86-93). Although the occurrence and consequences of aging-related aneuploidy in oocytes of humans and animal models have been extensively studied (Tarín et al., Biol Reprod 2001 65:141-150; Pan et al., Dev Biol 2008 316:397-407; Duncan et al., Biol Reprod 2009 81:768-776), approaches to maintain fidelity of chromosome segregation during meiotic cell division with age have remained elusive. At present there is no known intervention to improve the pregnancy outcome of older female patients. In animal studies, chronic administration of pharmacologic doses of anti-oxidants during the juvenile period and throughout adult reproductive life has been reported to improve oocyte quality in aging female mice (Tarín et al., Mol Reprod Dev 2002 61:385-397). However, this approach has significant long-term negative effects on ovarian and uterine function, leading to higher fetal death and resorptions as well as decreased litter frequency and size in treated animals (Tarín et al., Theriogenology 2002 57:1539-1550). Thus, clinical translation of chronic anti-oxidant therapy throughout reproductive life for maintaining or improving oocyte quality in aging females is impractical.
Mitochondrial dysfunction has a major role in reproductive senescence and, therefore, reproductive function in older women might be improved by the use of mitochondrial nutrients (Bentov et al., Fertil Steril 2010 93:272-275). Aging and age-related pathologies are frequently associated with loss of mitochondrial function, due to decreased mitochondrial numbers (biogenesis and mitophagy), increased aggregation of mitochondria, diminished mitochondrial activity (production of ATP, which is the main source of energy for cells) and mitochondrial membrane potential and/or accumulation of mitochondrial DNA (mtDNA) mutations and deletions. As oocytes age and oocyte mitochondrial energy production decreases, many of the critical processes of oocyte maturation required to produce a competent egg, especially nuclear spindle activity and chromosomal segregation, become impaired (Bartmann et al., J. Assist Reprod Genet 2004 21:79-83; Wilding et al., Zygote 2005 13:317-23). Nicotinamide adenine dinucleotide (NAD+) is a small molecule regulator of many other processes including signaling pathways, cell-cell communication, and epigenetic changes. Once thought to be very stable, levels of NAD+ rise in response to dieting and exercise. Increased NAD+ levels are also associated with the diet known as caloric restriction (CR), which is known to delay numerous aspects of aging and diseases, including infertility (Sinclair Mech Ageing Dev 2005 26:987; Selesniemi et al. Aging Cell 7:622-629, 2008).
NAD+ levels are important for the proper function of mitochondria and the cells that contain them. Cells with low mitochondrial NAD+ are prone to cell dysfunction and death (Yang et al., Cell 2008). Obesity and aging both reduce mitochondrial NAD+ levels, resulting in decreased mitochondrial function, increased cell death, and an acceleration of age-related diseases (Hafner et al. Aging 2010 2:1-10). As oocytes age and oocyte mitochondrial energy production decreases, many of the processes of oocyte maturation, especially meiotic spindle activity and chromosomal segregation, become impaired (Bartmann et al., J Assist Reprod Genet 2004 21:79-83; Wilding et al., Zygote 2005 13:317-23). Raising NAD+ levels is a viable option for increasing the bioenergetics and viability of cells, organs, tissues, and embryonic development. Downstream mediators include the sirtuin deaceylases (SIRT1-7) and the poly-ADP ribose polymerases (PARPs). It is known to those skilled in the art that increasing NAD+ levels and boosting mitochondrial function can mimic the health benefits of caloric restriction (Yang et al., Exp Gerontol 2006 41: 718-726).
The link between chronic anti-oxidant therapy for maintaining oocyte quality in females of advanced reproductive age is established (Tarín et al., Hum Reprod 1995 10:1563-1565) and data supporting a key role for mitochondrial dysfunction in eggs as a driving force behind age-related fertility problems are available. For example, experimentally-induced oxidative stress in isolated mouse oocytes reduces ATP levels, which increases meiotic spindle abnormalities leading to chromosomal misalignment (Zhang et al., Cell Res 2006 16:841-850). Additionally, while meiotic maturation of human oocytes can proceed over a range of ATP concentrations, oocytes with a higher ATP content show a much greater potential for successful embryogenesis, implantation and development (Van Blerkom et al., Hum Reprod 1995 10:415-424).
Along these same lines, heterologous transfer of cytoplasmic extracts from young donor oocytes (viz. obtained from different women) into the oocytes of older women with a history of reproductive failure, a procedure known as ooplasmic transplantation or ooplasmic transfer, demonstrated improved embryo development and delivery of live offspring. Unfortunately, however, the children born following this procedure exhibit mitochondrial heteroplasmy or the presence of mitochondria from two different sources (Cohen et al., Mod Hum Reprod 1998 4:269-280; Barritt et al., Hum Reprod 2001 16:513-516; Muggleton-Harris et al., Nature 1982 299:460-462; Harvey et al., Curr Top Dev Biol 2007 77:229-249). This is consistent with the fact that maternally-derived mitochondria present in the egg are used to “seed” the embryo with mitochondria, as paternally-derived mitochondria from the sperm are destroyed shortly after fertilization (Sutovsky et al., Biol Reprod 2000 63:582-590). Although the procedure involves transfer of cytoplasm and not purified mitochondria from the donor eggs, the presence of donor mitochondria in the transferred cytoplasm, confirmed by the passage of “foreign” mitochondria into the offspring, is widely believed to be the reason why heterologous ooplasmic transfer provides a fertility benefit (Harvey et al. Curr Top Dev Biol 2007 77:229-249). Irrespective, the health impact of induced mitochondrial heteroplasmy in these children is as yet unknown; however, it has been demonstrated that a mouse model of mitochondrial heteroplasmy produces a phenotype consistent with metabolic syndrome (Acton et al., Biol Reprod 2007 77: 569-576). Arguably, the most significant issue with heterologous ooplasmic transfer is tied to the fact that mitochondria also contain genetic material that is distinct from nuclear genes contributed by the biological mother and biological father. Accordingly, the children conceived following this procedure have three genetic parents (biological mother, biological father, egg donor), and thus represent an example of genetic manipulation of the human germline for the generation of embryos. Ooplasmic transplantation procedures that result in mitochondrial heteroplasmy are therefore now regulated and largely prohibited by the FDA. For details, see CBER 2002 Meeting Documents, Biological Response Modifiers Advisory Committee minutes from May 9, 2002, which are publically available from the FDA and “Letter to Sponsors/Researchers—Human Cells Used in Therapy Involving the Transfer of Genetic Material By Means Other Than the Union of Gamete Nuclei”, which is also publically available from the FDA on the worldwide web. While use of autologous mitochondria from somatic cells would avoid mitochondrial heteroplasmy, the somatic mitochondria are nonetheless inadequate, as they are prone to mitochondrial DNA damage and deletions resulting in heritable mutations. Autologous sources of female germ cells, namely OSCs and compositions obtained thereof (e.g., OSC cytoplasm or isolated mitochondria), in ooplasmic transplantation procedures would prevent mitochondrial heteroplasmy, and alleviate ethical and safety concerns currently associated with the procedure. Importantly, oocytes, which are prone to aging-associated defects, are not of high enough quantity or quality to be reliably used in such procedures.
Accordingly, it is desirable to restore the quality of aged oocytes, as well as to further enhance OSCs or improve derivatives thereof (e.g., cytoplasm or isolated mitochondria) for use in conducting a range of assisted reproductive technologies.